The present invention relates to microorganisms of novel species and methods for using the same. More specifically, the present invention relates to microorganisms of novel species belonging to the genus Bacillus and methods for using the same.
Mankind has utilized various microorganisms to date. For example, a wide variety of foods such as alcoholic drinks, fermented seasonings (e.g. soybean paste and soy sauce), fermented dairy products (e.g. cheese and yogurt) and bread have been produced using actions of microorganisms. In addition, microorganisms have also been utilized in the manufacturing of pharmaceuticals and agricultural chemicals, in the production of energy sources such as alcohol and methane, in the treatment of waste and wastewater, and so forth.
The technology of using functions of living things (especially microorganisms) in industrial processes, which is called biotechnology, is developing remarkably in these days. The discovery of novel, useful microorganisms contributes to the advance of biotechnology.
Under circumstances, it is an object of the present invention to provide novel, useful microorganisms.
It is another object of the present invention to provide methods of use of the microorganisms.
The present inventors have isolated from soil a microorganism of a novel species having a smell of coffee. Thus, the present invention has been achieved. The present invention provides microorganisms belonging to the genus Bacillus that are capable of reducing nitrates and contain chitin and/or chitosan in their cell walls. The microorganism of the invention may have a smell of coffee. The microorganism of the invention may be a microorganism belonging to Bacillus subtilis. As one example of the microorganism of the invention, Bacillus subtilis takemi (FERM BP-6589) may be given.
The present invention also provides a method for improving soil using the microorganism, and a composition for improving soil comprising the microorganism.
Further, the present invention provides a method for treating organic waste by fermentation using the microorganism, and a composition for treating organic waste by fermentation comprising the microorganism.
Further, the present invention provides a method for fermenting soybeans using the microorganism, and soybeans that have been fermented using the microorganism.
The present invention also provides a method of using the microorganism as a feed additive, and a feed additive comprising the microorganism.
The present invention also provides a method of using the microorganism for reducing bitterness, and a composition for reducing bitterness comprising the microorganism. The present invention also provides a method of using the microorganism as a food additive, and a food additive comprising the microorganism.
The present invention also provides a method for inhibiting bacterial growth using the microorganism, and an anti-microbial composition comprising the microorganism. Specific examples of target bacteria of which the growth can be inhibited by the microorganism of the invention include staphylococci, pathogenic E. coli O157 and O147, dermatophytes, and bacteria of the family micrococcaceae.
The microorganism of the invention is a bacterium belonging to the genus Bacillus that is capable of reducing nitrates and contains chitin and/or chitosan in its cell walls. The microorganism of the invention may have a smell of coffee. As a specific example of such a microorganism, Bacillus subtilis takemi (FERM BP-6589) may be given. This microorganism has been isolated as described below from a soil sample collected in Siberia.
Briefly, 500 xcexcl of sterilized distilled water was added to 50 mg of a Siberian soil sample, which was then shaken for 30 min to prepare a suspension (presenting a brown color). Using this suspension as a stock solution, 10-2-10-7 dilutions were prepared by 10-fold serial dilution. Then, 100 xcexcl of each dilution was plated on PDAYC medium (Potato Dextrose Agar medium containing Yeast Extract and Casion) and spread uniformly with a spreader, followed by stationary culture under the condition of all day light and at 20xc2x0 C. As a result, formation of white colonies was observed in 10xe2x88x922xe2x88x9210xe2x88x924 dilutions. It was found that these colonies were not filamentous fungi as a result of microscopic examination. Since all the colonies were white, it was presumed that a single species was growing there. Formation of no colonies was observed in dilutions below 10xe2x88x924.
The characters of the isolated microorganism were as summarized below.
It was also found that the GC content in the intracellular DNA (as determined by HPLC) was 46% by mol.
Based on the results of the morphological observation, physiological character tests and the determination of the GC content in the intracellular DNA so far described, the microorganism was identified referring to Gordon, R. E., Haynes, W. C. and Pang, C. H., xe2x80x9cThe Genus Bacillusxe2x80x9d (1973), U.S. Department of Agriculture and Sneath, P.H.A., Mair, N. S., Sharpe, M. E. and Holt, J. G., xe2x80x9cBergey""s Manual of Systematic Bacteriologyxe2x80x9d Vol. 2, (1986) Williams and Wilkins. As a result, the soil bacterium was identified as a bacterial species belonging to Bacillus subtilis. This bacterium was designated Bacillus subtilis takemi and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 Higashi 1-Chome, Tsukuba City, Ibaraki Pref., Japan) on Dec. 1, 1998 under the accession number FERM BP-6589. According to the terms of the deposit at the National Institute of Bioscience and Human Technology, all restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. Bacillus is a group of gram-positive, rod-shaped bacteria that form thermo-stable spores and are distributed widely in common environment such as soil. Bacillus subtilis is also known as hay bacillus. In addition to the above-described characters, Bacillus subtilis takemi was confirmed to have a remarkable smell of coffee when culture plates of B. subtilis takemi cultured as described above were kept in a refrigerator adjusted at about 5xc2x0 C. Further, it was confirmed that Bacillus subtilis takemi contains chitin and/or chitosan in its cell walls.
It should be noted that, not to mention natural and artificial mutants of Bacillus subtilis takemi, all of those microorganisms of the genus Bacillus that are capable of reducing nitrates and contain chitin and/or chitosan in their cell walls are included in the present invention.
The microorganism of the invention has preferable properties as a soil microorganism. That is, the microorganism of the invention can reduce nitrates. Also, the chitin matter contained in its cell walls is degraded to chitosan/chitosan oligosaccharides, which in turn react with toxins generated by harmful microorganisms to thereby inhibit the transcription of their DNA to RNA. As a result, the growth of harmful microorganisms is prevented, and damage caused by continuous cropping is reduced. Further, it is convenient to use the microorganism of the invention since its concentration can be judged with its smell of coffee. Therefore, it is possible to improve soil using the microorganism of the invention.
Briefly, the microorganism of the invention and optionally other microorganisms effective for soil improvement are added to an aqueous solution containing nutrients suitable for the growth of the microorganisms. Further, a carrier is added to this solution, which is then agitated. By this operation, the microorganism of the invention is carried by the carrier. The resultant dispersion mixture is maintained within the above-described temperature range appropriate for the growth of the microorganism (e.g. 25-45xc2x0 C.) to thereby grow the microorganism.
Specific examples of nutrients suitable for the growth of the microorganism of the invention include, but are not limited to, carbon sources such as starch, dextrin, glycerol, glucose, sucrose, galactose, inositol and mannitol; and nitrogen sources such as peptone, soybean powder, meat extract, rice bran, wheat-gluten bread, urea, corn steep liquor, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds. Further, inorganic salts such as table salt, phosphates, metal salts of potassium, calcium, zinc, manganese, iron, etc. may be appropriately added. If necessary, animal oil, vegetable oil, mineral oil or the like may be added as a defoaming agent. A commercially available medium such as Potato Dextrose Agar Medium (product name: NISSUI 05707) may be used. Specific examples of other microorganism effective for soil improvement include, but are not limited to, hydrolase producing microorganisms belonging to the genus Bacillus, Lactobacillus or the like; yeast species belonging to the genus Saccharomyces, Tolura or the like; and filamentous fungi belonging to the genus Aspergillus, Rhizopus or the like. Specific examples of the carrier on which the microorganism of the invention is to be carried include, but are not limited to, porous ceramics, wood pieces, charcoal, straw pieces, and other porous materials on which the microorganism of the invention can survive. The appropriate number of the microorganism cells of the invention per gram of the carrier is 102xe2x88x921010, preferably 103xe2x88x92106.
A carrier carrying the microorganism of the invention or powder of such a carrier has a function as a soil improving material by itself. However, usually, such a carrier or powder thereof is mixed with raw materials of an organic fertilizer and fermented so that the resultant mixture has both fertilizer effects and soil improvement effects. The carrier carrying the microorganism of the invention or powder thereof may be mixed with organic fertilizer materials at a rate of 2-10 g/kg. For example, powder of a porous ceramic on which the microorganism of the invention has been grown is mixed with bark, poultry droppings and other organic fertilizer materials and then fermented for about 30 days. As the fermentation of the organic materials proceeds, a composition for soil improvement with a high fertilizer response property is obtained which contains microorganisms effective for soil improvement and yet which is rich in chitin and/or chitosan effective as a soil-improving material.
In the application of the above-described composition for soil improvement to soil, 150-600 kg, preferably 220-520 kg of the composition comprising the microorganism of the invention may be applied per 10 a. of farmland (1 a.=100 m2). The composition may be applied at any time from the early growing stage of plants to the harvest stage. The frequency of application is not particularly limited. The time and frequency of the application may be appropriately selected depending on the state of soil and the kind of crops.
It is also possible to treat organic waste by fermentation using the microorganism of the invention. The term xe2x80x9corganic wastexe2x80x9d used herein refers to waste comprising organic matter, which may be domestic waste, industrial waste or any other waste comprising organic matter. Poultry droppings, cattle droppings and food sludge are also included in this term. Microorganisms are involved in the fermentation and degradation of organic matter. During these processes, bad smells are generated by degradation and by putrefactive microorganisms. Further, offensive odor caused by the growth of putrefactive microorganisms as a result of unbalanced oxygen supply in the fermentation of domestic organic waste, cattle droppings, poultry droppings and food sludge by aerobic bacteria has become a big social problem throughout the country. When organic waste is treated by fermentation using the microorganism of the invention, bad smells can be prevented. Thus, the microorganism of the invention greatly improves the environment. Further, the organic waste that has been treated by fermentation using the microorganism of the invention can be recycled as an organic fertilizer of a high quality.
Preferably, the microorganism of the invention is used in combination with conventional microorganisms used in waste treatment (mainly, aerobic bacilli).
As described above, a carrier (e.g. wood pieces, porous ceramic beads, grains or granules) carrying the microorganism of the invention is prepared and then mixed with organic waste for the purpose of fermentation treatment. The carrier carrying the microorganism of the invention may be used at 2-10 grams per kilogram of organic waste. Specific examples of waste treating microorganisms that may be used in combination with the microorganism of the invention include, but are not limited to, bacteria and filamentous fungi that degrade saccharides contained in waste; aerobic actinomycetes that degrade hemicellulose in waste; anaerobic bacteria that degrade cellulose in waste; and aerobic basidiomycetes that degrade lignin. During the fermentation treatment, it is preferable to provide aeration a rate of 100-200 ml/min per liter of organic waste. Alternatively, air may be supplied by agitation instead of aeration. The temperature may be ambient temperature or may be adjusted at 12-51xc2x0 C., preferably 25-45xc2x0 C. In order to improve moisture adjustment and aeration efficiency, auxiliary materials such as wood chips and rice hulls may be added. The period of fermentation treatment varies depending on the composition of the waste to be treated. Usually, this period is 1-30 days, preferably 6-20 days. It is even possible to annihilate organic waste when the above-described fermentation is enhanced by adding a fermentation medium to the waste. If organic waste is considered as a resource and its recovery as a well-balanced fertilizer is desired, the composition of the organic waste may be adjusted by adding thereto cattle droppings, residues from the processing of animal meat or fish meat, etc. The thus treated organic waste becomes an organic fertilizer of a high quality since it comprises chitin and/or chitosan contained in microorganisms.
The results of homology analysis between the 16S ribosomal RNA (16S rRNA) of Bacillus subtilis takemi and that of Bacillus subtilis nattoh revealed about 93% homology (FIG. 1). Bacillus subtilis nattoh is used in the manufacturing of nattoh (fermented soybeans). Nattoh (i.e. fermented soybeans) is known to have an action of lysing clotted blood and, thus, is highly evaluated as a health food. Further, chitin, chitosan or chitosan oligosaccharides, which are metabolites of Bacillus subtilis nattoh, are known to play an important role to enhance immunocompetence. However, many people dislike nattoh since it emits an odor of nitrates. Also, production of nitrates results in deterioration of proteins. The microorganisms of the invention including Bacillus subtilis takemi have a nitrate-reducing action. Therefore, when soybeans are fermented using the microorganism of the invention, generation of the odor and deterioration of proteins can be prevented. Conventional nattoh has been served as a side dish to cooked rice or the like. On the other hand, the nattoh prepared with the microorganism of the invention has a smell of coffee, and can be taken with bread or used in sandwiches or other western style dishes. As a result, it will become possible to spread nattoh throughout the world as a functional food that is highly nutrient and effective for not only the maintenance of health but also the promotion of health.
Nattoh can be prepared using the microorganism of the invention instead of conventional Bacillus subtilis nattoh. Briefly, the microorganism of the invention is added to boiled soybeans and cultured for growth. After the microorganism performed fermentation action, the soybeans are ripened. Methods for preparing nattoh are well known and described, for example, in Susumu Tsuji, xe2x80x9cFood Processing Technology Handbook: Revised Editionxe2x80x9d, pp. 138-143, KENKIN-SHA (1971).
As one application of the above-described method, catechin and ginkgo leaf extract are added to soybeans during the process of preparation of nattoh using the microorganism of the invention. Nattohkinase that lyses clotted blood has an effect of preventing the generation of thrombi or the like; catechin that is anti-microbial has an effect of inhibiting viral growth; and ginkgo leaf extract that is an SOD-like antioxidant has an effect of preventing the generation of active oxygen and improving blood flow in the blood capillary. Since the microorganism of the invention is a nitrate-reducing bacterium, the resultant nattoh has only slight smell of nattoh derived from the odor of ammonium or the like, and yet this slight smell is camouflaged by a smell of coffee. Thus, the resultant nattoh is easy to eat. Besides, an effect of rendering blood vessels strong and an effect of balancing cholesterols in blood vessels that are attributable to the above-described effects can be added to the nattoh. Thus, a completely novel nattoh can be prepared which is effective in preventing diseases of adult people and senile dementia. Although catechin and ginkgo leaf extract have bitterness, the mild taste of the microorganism of the invention reduces their bitterness. Further, it is believed that the microorganism emitting a smell of coffee camouflages their bitterness and the smell peculiar to nattoh.
It is also possible to use the microorganism of the invention as a feed additive. Specific examples of feeds to which the microorganism of the invention may be added include, but are not limited to, feed for domestic animals, fish feed and pet food. For example, when the microorganism of the invention is added to feed for domestic animals, physiological disorders of the animals fed with such feed can be alleviated by the action of the nitrate reductase contained in the microorganism. Besides, the feed conversion ratio in these animals is increased to thereby decrease the calorie of proteins discharged into their droppings. As a result, the offensive odor of droppings is alleviated.
The microorganism of the invention may be added to feed at a rate of 103xe2x88x92106 cells/g.
It is also possible to use the microorganism of the invention to reduce bitterness of foods, pharmaceuticals, cosmetics and the like. Specific examples of foods having bitterness include, but are not limited to, citrus fruits such as grapefruit, lemon, and juice thereof; vegetables such as tomato, green pepper, celery, and juice thereof; soybean products such as nattoh and soybean milk; fish meat and processed marine products such as ground fish meat; and meat and processed meat products. Specific examples of pharmaceuticals having bitterness include, but are not limited to, catechin and ginkgo leaf extract. Specific examples of cosmetics having bitterness include, but are not limited to, cosmetics applied to the face and cosmetics applied to the oral cavity.
It is contemplated that the microorganism of the invention can be applied in various ways as a food additive. The term xe2x80x9cfoodxe2x80x9d used herein includes any and all foods and drinks. Foods to which the microorganism of the invention may be added are not particularly limited. The microorganism may be added, for example, to livestock meat, milk, fishes and shellfishes, and processed products thereof; and cereals, beans, potatoes, vegetables, fruits, and processed products thereof. For example, the microorganism of the invention may be used as a food additive for the preparation of ice cream or yogurt with a smell of coffee. The food additive consisting of the microorganism of the invention has a smell of coffee but contains no caffeine. Thus, the additive has an advantage that it does not form a habit. The microorganism of the invention may be added to food at a rate of 103xe2x88x92106 cells/g. However, this range may vary depending on the type of food and other factors.
When the microorganism of the invention is coated on a plastic wrapping sheet or inner surfaces of a plastic food container, food wrapped therewith or contained therein can be preserved for a longer period of time without harmful effect on the it, because putrefaction and growth of miscellaneous microorganisms are prevented by the anti-microbial property of chitin and chitosan. The microorganism of the invention may be coated at a rate of 101xe2x88x92105 cells/cm2 of wrapping sheet or food container. For the coating, live or dead cells of the microorganism of the invention are dispersed in pure water and disrupted by sonication. Then, the resultant dispersion liquid is coated thinly on a wrapping sheet or food container.
In inflammatory sites of patients with atopic dermatitis, staphylococci are growing. It is known that when a paste containing Bacillus subtilis nattoh is applied to these sites, B. subtilis nattoh eats staphylococci to thereby inhibit the advance of purulence and/or atopy. When the microorganism of the invention is used instead of B. subtilis nattoh, the growth of staphylococci is inhibited by the effect of chitin and chitosan in addition to the conventional effect of B. subtilis nattoh. Thus, therapeutic effect on atopy is further enhanced. The microorganism of the invention may be formulated into various formulations such as ointment, lotion, cream, paste, gel, emulsion and pack. The content of the microorganism of the invention in such a formulation is not particularly limited; it may be contained at 0.0001-0.1% by weight. The formulation containing the microorganism of the invention may be applied to inflammatory sites of atopic dermatitis patients in a dosage of 10xe2x88x926xe2x88x9210xe2x88x923 g (in terms of the microorganism that is the active ingredient) one to several times a day. In the preparation of these formulations, hydrocarbons such as squalane, paraffin and vaseline; oils such as olive oil and almond oil; waxes such as beeswax and lanolin; fatty acids such as stearic acid and oleic acid; higher alcohols such as cetanol and stearyl alcohol; synthetic esters such as glycerol triester; oil phase components such as silicone oil; moisture retentives such as glycerol, propylene glycol, sorbitol and polyethylene glycol; thickning agents such as quince seed gum, pectin and cellulose derivatives; alcohols such as ethanol and isopropyl alcohol; purified water such as deionized water; surfactants such as glycerol monostearate, sorbitan fatty acid ester, fatty acid soap and sodium alkylsulfate; alkali such as potassium hydroxide, sodium hydroxide and triethanolamine; flavoring agents; pigments; chelating agents; preservatives; anti-oxidants; buffers; vitamins; UV absorbents; amino acids and the like may be combined.